In recent years, it has been reported that various substances having sex hormone-like activity occur in the environment, and the problem of environmental pollution by such substances has been becoming more and more serious. The substances having sex hormone-like activity are chemical substances acting as endocrine disruptors exerting an influence on biogenic hormones. Typical examples of such substances include substances affecting the functions of estrogen and androgen, namely sex hormone-like substances such as estrogen-like acting substances, antiestrogen-like acting substances, androgen-like acting substances and antiandrogen-like acting substances.
There is growing apprehension that such sex hormone-like substances will destroy the hormonal balance in animals and disturb the ecosystem or cause various diseases. Therefore, the advent of means for screening out in a simple and accurate manner such sex hormone-like substances from among the existing chemical substances and the chemical substances that will be newly developed in the future is now demanded in the relevant industries.
Currently known as the means for detecting sex hormone-like substances are the reporter gene assay methods which utilize an estrogen receptor gene or androgen receptor gene (cf. e.g. Japanese Unexamined Patent Publication Nos. 2000-300258 and 2000-300259). For the method utilizing the androgen receptor gene, for instance, the principle of the assay method is as follows.
Thus, when the androgen receptor (AR) gene-expressing cells with a reporter gene (e.g. luciferase (Luc) gene) introduced therein so that the expression of the reporter gene can be controlled by the transcriptional regulatory region having an androgen-response elements (ARE) (such cells are hereinafter sometimes referred to as “luciferase-expressing cell”) are brought into contact with an androgen-like acting substance or antiandrogen-like acting substance as a test substance, this test substance binds to the AR gene occurring in the cell and activates the androgen receptor. As a result of this activation, the transcription of the reporter gene is initiated and the transcription product (reporter protein, e.g. Luc) is produced. Therefore, the expression of the AR gene can be estimated by taking the expression level or activity of this transcription product as an indication, hence the androgen-like activity or antiandrogen-like activity of the test substance can be estimated.
In cases where, in the assay method using the above luciferase-expressing cells is used, a substance having an antagonistic activity against the androgen receptor (antiandrogen activity) is used as the test substance and when a fixed amount of an agonist is added to the assay system in advance, the inhibitory activity of the test substance against that agonist can be evaluated in terms of the decrease in the expression level or activity of luciferase as a reporter protein.
However, since this assay system uses living cells, not only the antagonist activity intrinsic in the test substance but also a toxicity (e.g. cytotoxicity) of the test substance, for instance, presumably causes the decrease in reporter protein activity. That is to say, the substance to be tested is by nature toxic to cells and, as the concentration thereof increases, the cytotoxicity thereof increases, resulting in hypofunction of the cells themselves, hence in a reduced level of reporter protein expression. Therefore, this assay system has a fatal defect in that the cytotoxicity or like activity of the test substance counterbalances the antagonistic activity of the test substance, which is to be assayed, and thus makes it difficult to accurately detect and assay that activity. Similarly, when the test substance is a substance having an agonistic activity, the cytotoxicity or like activity of the test substance itself renders it difficult to accurately detect and assay the agonist activity desired to be known, in the same manner as mentioned above.
In the art, there is no means for precisely knowing the correlation between the sex hormone-like activity of such a test substance and such a factor as cytotoxicity that may lead to erroneous judgments of the detection results, and no means has been developed for properly evaluating test substances for sex hormone-like activity while neutralizing the effects of such factors. Thus, there is no indicator known in the art that can accurately reflect the cellular hypofunction (reduction in activity), in particular the reduction in cellular capacity for protein expression, due to the cytotoxicity or like activity of the test substance. Of course, there is no technology developed for assaying the sex hormone-like activity of a test substance utilizing such indicator.
Meanwhile, technologies of counting viable cells are known in the art [e.g. MTT method (cf. e.g. Molecular and Cellular Endocrinology, 160, (2000), pp. 39-49) and cell fluorescence labeling method using AlamarBlue (cf. e.g. Toxicology and Applied Pharmacology, 155, 150-160 (1999)). However, these technologies merely serve as means for judgment about life or death of cells. They can never make it possible to accurately assay and judge about such cellular hypofunction as mentioned above, in particular a reduced protein-expressing capacity. Even if these technologies are combined with the above-discussed reporter gene assay methods, it will be impossible to perform accurate sex hormone-like activity assaying by subtracting the cellular hypofunction-due errors from the values measured in the reporter gene assay systems.
An object of the present invention is to provide an improved bioassay method by which the sex hormone-like activity inherent in a test substance can be accurately assayed and judged by accurately estimating the test substance-due hypofunction of cells used in this type of reporter gene assay system, namely the variation in cellular protein expression activity.